CN103442732B - Nucleic acid molecule encoding hepatitis B virus core protein and vaccine comprising the same - Google Patents

Abstract

Provided herein are nucleic acid sequences that encode novel consensus amino acid sequence of HBV core protein, as well as genetic constructs/vectors and vaccines that express said protein sequences. Also provided herein are methods for generating an immune response against HBV using the nucleic acid sequences that are provided.

Description

The nucleic acid molecules of encoding hepatitis B viral core protein and the vaccine comprising which

Invention field

The present invention relates to the nucleotide sequence of encoding hepatitis B virus (HBV) core protein and its fragment；It is related to B-mode liver Scorching virus (HBV) core protein and its fragment, the improvement of the immunoreation for being related to improved HBV vaccines, HBV being directed to for induction Method and the individual improved method for HBV immunity is made for pre- defense sector and/or treatment.

Background of invention

This application claims the priority of the U.S. Provisional Patent Application No. 61/442,162 submitted to on 2 11st, 2011, institute State patent application to be herein incorporated by reference.

Hepatitis B is common infection popular in global range, which results in liver cirrhosis, liver failure and liver thin The development of born of the same parents' cancer.Significant amount of hepatitis cases are not reported out and are because the asymptomatic characteristic of the disease.Even so, About 3.5 hundred million chronic hepatitis b disease examples are reported still every year.The crowd of most of virus infection is in undeveloped country or sends out Country in exhibition.

It is described virus be based on epitope present on its envelope protein and be divided into four kinds of predominant serotypes (adr, adw, ayr、ayw).According to the change of genome sequence, there are at least eight kinds genotype (A-H) in HBV.The replacement genotype of HBV has Popular geographical distribution.

Table 1 is the geographical distribution of HBV gene type.

The geographical distribution of table 1-HBV

Medical virology magazine (J.Med.Virol), DOI 10.1002jmv

HBV gene group is circular DNA molecule, and the molecule is mainly double-strand but the double-strand is with by longer than another chain The single-stranded regions that one chain is produced.The double-stranded region by about 3020 nucleotide relatively short chain a chain and about 3320 nucleoside The longer chain of acid hybridizes and produces.Single-stranded regions on the non-hybridising nucleotides of the longer chain and HBV archaeal dna polymerase phases Associate.Both HBV gene group DNA and HBVDNA polymerases are included in and are formed by multiple HBcAgs (HBcAg) molecule In nucleocapsid.HBcAg is by HBsAg matter (HBsAgs) and lipid molecular peplos.

The HBV gene group contains four open reading frames (ORF)：1) ORF of encoding HBV DNA polymerase, 2) has The ORF of two start codons, wherein being connected to the sequential coding core protein of second start codon and including another The sequential coding of individual upstream start codon is referred to as the sequence of pre-C；3) ORF with three start codons, wherein Individual coded surface protein (gp27), the upstream start codon of a sequence for being referred to as pre-S2 (gp36) including coding, And another includes that coding is referred to as the start codon of the more upstream of the sequence of pre-S1 (gp42)；And 4) encode The ORF of HBxAg, the HBxAg are the less protein being appreciated that of function.

Vaccine and treatment for HBV infection is related to inject the subvirus from purification in the blood plasma of chronic carriers Grain or the subviral particle being generated as recombinant protein in the eukaryotic cell lines of stable transfection.The subviral particle It is virus protein, and these vaccines is commonly known as subunit vaccine.HBV albumen is administered to individuality and is changed into individuality Immune target.In the individuality being uninfected by, for subunit vaccine immunoreation protection described in be uninfected by individuality and exempt from By HBV infection.In infected individuals, there can be therapeutic effect by vaccine-induced immunoreation.

Qi Sali F.V. (Chisari F.V.), American Journal of Pathology (Am J Pathol.), 2000.156: World virusology such as 1117-1132 and Pa Basi P. (Pumpeus P.) (Intervirology) 2001.44:98-114 is public HBV gene group structure is opened.Enlightening how P. (Deny P.) and F. Zou Li (F.Zoulim), pathology and biology (Pathologie Biologie) 2010, August, 58 (4):245 53 diagnosis and treatment for discussing hepatitis B viruss.Wheat Gram M.L. (Michel M.L.) and P. base of a fruit Aurions (P.Tiollais), pathology and biology 2010, August, 58 (4): 288 95 discuss hepatitis B vaccine and their protection effect and treatment potential.PCT Publication W02004026899 is disclosed Immunogenic purposes containing the peptide sequence with HBV aminoacid sequences.The application W02008093976 of PCT Publication is disclosed Include HBV coded sequences, protein and the vaccine of the vaccine comprising recombinant full-lenght HBsAg and HbcAg.It is whole Individual HBsAg is made up of the surface protein (L albumen, M albumen and S protein) of three types.The application of PCT Publication W02009130588 discloses HBV coded sequences, protein and the epidemic disease of the nucleic acid including encoding hepatitis B virus core antigen Seedling, the nucleic acid are optimized to the codon for expressing in people.PCT Publication W02010127115 is disclosed using restructuring Vehicle delivery HBV sequences.

Available HBV vaccines have shown some effects, but produce expensive.Additionally, from the subunit of blood plasma Vaccine is also with the worry in terms of safety.Some vaccine approach are had studied, methods described includes living based on restructuring Those methods of the DNA vaccination of carrier, synthetic peptide and the codon optimization coded sequence comprising HBV protein.These other Method so far have change limited efficacy.Further, since the difference of genome, some HBV vaccines are on some ground Positive effect is showed in reason region and limited effect is showed in other regions.

Studied direct administration of nucleic acid sequence vaccination to be carried out for animal and human disease, and much made great efforts collection In in effective and efficient means of delivery of nucleic acids, to produce the necessary expression of required antigen, so as to cause immunogenicity Reaction and the success of final this technology.

DNA vaccination allows endogenous antigen to synthesize, and this induces the cell toxicant that the I classes of the complexity of CD8+ histocompatibilitys are limited Property T lymphocytes, the lymphocyte subunit vaccine are seldom obtained.Additionally, the antigen occurred in one section of continuous period of time Synthesis can aid in the needs for overcoming low-response and removing or reduce reinforcing injection.In addition, DNA vaccination seems highly stable And produce simple.Furthermore, it is possible to pass through with reference to strategy such as codon optimization, RNA optimizations and add immunoglobulin leader sequence To induce wider cell immune response.

DNA vaccination is safe and stable, be readily produced, and the well-tolerated in people, wherein preclinical laboratory indicate almost do not have There are plasmid integration sign [Martin T. (Martin, T.) etc., plasmid DNA malaria vaccine：Genome conformity after intramuscular injection Probability.(Plasmid DNA malaria vaccine:the potential for genomic integration After intramuscular injection.) human gene therapy (Hum Gene Ther), 1999.10 (5):759-68 Page；Nichols W.W. (Nichols, W.W.) etc., possible DNA vaccination is incorporated in host cell gene group. (Potential DNA vaccine integration into host cell genome.) NYAS science annual report (Ann N Y Acad Sci), 1995.772:30-9 page].Additionally, DNA vaccination is highly suitable for repetitive administration, this is because Effect of the fact, i.e. vaccine is not affected [Chang Tegu M. by the pre-existing antibody titer on carrier (Chattergoon, M.), J. pool Ilyushin (J.Boyer) and D.B. wieners (D.B.Weiner), genetic immunization：Vaccine and The New Times of immunization therapy.(Genetic immunization:a new era in vaccines and immune Therapeutics.) U.S. experimental biology community's magazine of association (FASEB J), 1997.11 (10):753-63 page].So And, for the immunogenicity for using a major obstacle of DNA vaccination being clinically the platform when moving to larger animal [Liu M.A. (Liu, M.A.) and J.B. ells are silent (J.B.Ulmer), people's clinical experiment of Plasmid DNA vaccines for reduction. (Human clinical trials of plasmid DNA vaccines.) hereditism is in progress (Adv Genet), 2005.55:25-40 page].

Nearest technological progress in terms of the immunogenic engineering of DNA vaccination has improved the expression of DNA vaccination and has exempted from Epidemic focus, such as codon optimization, the RNA optimization of these technologies and the addition of immunoglobulin leader sequence [the strong S. of peace moral (Andre, S.) etc., by with optimizing codon using the increasing that carries out DNA vaccination inoculation and cause of synthesis gpl20 sequences Strong immunoreation.(Increased immune response elicited by DNA vaccination with a Synthetic gpl20 sequence with optimized codon usage.) Journal of Virology (J Virol), 1998.72(2):1497-503 page；Demi L. (Demi, L.) etc., codon is using optimization to encoding human immunodeficiency virus 1 The expression of the DNA candidate vaccines of type Gag albumen and immunogenic multiple impact.(Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines 1 Gag protein. of encoding the human immunodeficiency virus type) Journal of Virology, 2001.75(22):10991-1001 page；Thunder enlightening D.J. (Laddy, D.J.) etc., the new DNA vaccination pin based on consensus sequence Immunogenicity to bird flu.(Immunogenicity of novel consensus-based DNA vaccines Against avian influenza.) vaccine (Vaccine), 2007.25 (16):2984-9 page；Fu Lelan L. (Frelin, L.) etc., codon optimization and mRNA amplifications effectively enhance hepatitis c virus non structural 3/4A genes Immunogenicity.(Codon optimization and mRNA amplification effectively enhances the Immunogenicity of the hepatitis C virus nonstructural 3/4A gene.) gene therapy (Gene Ther), 2004.11 (6):522-33 page], and the technology of the nearest exploitation in plasmid delivery system aspects, it is such as electric [horizontal tail L.A. (Hirao, L.A.) etc. carries out Intradermal/subcutaneous inoculation by electroporation and improves in pig and Rhesus Macacus for perforation Plasmid vaccine is delivered and effect.(Intradermal/subcutaneous immunization by electroporation Improves plasmid vaccine delivery and potency in pigs and rhesus macaques.) epidemic disease Seedling, 2008.26 (3):440-8 page；Triumphant A. (Luckay, A.) etc. is drawn, Plasmid DNA vaccines design and In vivo electroporation are in perseverance The impact of the vaccine specific immunoreation produced in the monkey of river.(Effect of plasmid DNA vaccine design and in vivo electroporation on the resulting vaccine-specific immune Responses in rhesus macaques.) Journal of Virology, 2007.81 (10):5257-69 page；Allan G (Ahlen, G.) etc., In vivo electroporation is strengthened by increased partial dna intake, protein expression, inflammation and CD3+T cellular infiltrations The immunogenicity of hepatitis c virus non structural 3/4A DNA.(In vivo electroporation enhances the immunogenicity of hepatitis C virus nonstructural 3/4A DNA by increased local DNA uptake,protein expression,inflammation,and infiltration of CD3+T Cells.) Journal of Immunology (J Immunol), 2007.179 (7):4741-53 page].In vivo electroporation technology is in the clinical examination of people Test On.Additionally, research has shown to increase the wide of cell immune response compared with single native antigen using total immunogen General property [tight J. (Yan, J.) etc., the increasing caused by peplos DNA vaccination of the HIV-1B hypotypes of through engineering approaches based on consensus sequence Strong cell immune response.(Enhanced cellular immune responses elicited by an Engineered HIV-1 subtype B consensus-based envelope DNA vaccine.) cell therapy (Mol Ther), 2007.15 (2):411-21 page；Rowland M. (Rolland, M.) etc., people's immunity at the evolutionary system center of ancestors lack Fall into reconstruct and the function of viral 1 type protein.(Reconstruction and function of ancestral center- 1 proteins. of of-tree human immunodeficiency virus type) Journal of Virology, 2007.81 (16): 8507-14 page].

Still there is the nucleic acid construct to encoding HBV antigens and the combination of the immunoreation of HBV is directed to suitable for induction The needs of thing.Still exist to economy and be effectively directed to the needs of the effective vaccine of HBV.Still exist to increasing neutralizing antibody Level and cause T cell component effective vaccine needs.Still exist to the effective vaccine for HBV, including for tool There are the HBV Strain of extensive genotype scope effectively those vaccines, and effectively general epidemic disease preferably in global range The needs of Seedling.

Summary of the invention

An aspect of of the present present invention includes the vaccine suitable for induction for the immunoreation of HBV.With for several genes The HBV core specific antigens that the exploitation of the HBV immunization therapy vaccines of the extensive effect of type can be generally preserved based on targeting, make There is provided with the therapeutic DNA vaccine for HBV infection.Using total HBV immunogens induction of wider cellular immunization Reaction, and go for making the different degree of sequence between different virus strain reduce to minimum.

Provided herein is selected from the protein of group consisting of：Comprising SEQ ID NO:2 protein；With SEQ ID NO:295% homologous protein；SEQ ID NO:2 fragment；With SEQ ID NO:The homologous protein of 2 fragment 95%；SEQ ID NO:4, with SEQ ID NO:495% homologous protein；SEQ ID NO:4 fragment；With SEQ ID NO:4 fragment 95% Homologous protein；SEQ ID NO:6, with SEQ ID NO:695% homologous protein；SEQ ID NO:6 fragment；And With SEQ ID NO:The homologous protein of 6 fragment 95%.

The nucleic acid molecules of the sequence comprising coding one or more protein molecule mentioned above are also provided.In some realities Apply in scheme, the nucleic acid molecules include the sequence selected from group consisting of：SEQ ID NO:1；With SEQ ID NO: 195% homologous nucleotide sequence；SEQ ID NO:1 fragment；With SEQ ID NO:The homologous nucleotide sequence of 1 fragment 95%；SEQ ID NO:3；With SEQ ID NO:395% homologous nucleotide sequence；SEQ ID NO:3 fragment；With SEQ ID NO:3 fragment 95% homologous nucleotide sequence；SEQ ID NO:5；With SEQ ID NO:595% homologous nucleotide sequence；SEQ ID NO:5 piece Section；And with SEQ ID NO:The homologous nucleotide sequence of 5 fragment 95%.

The present invention some in terms of provide induction for the cAg from one or more HBV gene type immunoreation Method, the method comprising the steps of：Such nucleic acid molecules and/or compositionss are applied to individual.

The other aspect of the present invention provides the method for protecting individuals from HBV infection.These methods are comprised the following steps：It is right The individual nucleic acid molecules comprising such nucleotide sequence or compositionss for applying prevention effective dose；Wherein described nucleotide sequence exists Express in the individual cell, and react for the protein inducing protective immunity by the nucleic acid sequence encoding.

In terms of some of the present invention, there is provided for treatment by the method for the individuality of HBV infection.These methods include Following steps：To the individual such nucleic acid molecules and/or compositionss for applying therapeutically effective amount.

The aspect of the present invention is additionally related to the nucleic acid of the protein that group consisting of is selected from comprising protein or coding Vaccine：Comprising SEQ ID NO:2 protein, with SEQ ID NO:295% homologous protein；SEQ ID NO:2 piece Section；With SEQ ID NO:The homologous protein of 2 fragment 95%；SEQ ID NO:4, with SEQ ID NO:495% homologous albumen Matter；SEQ ID NO:4 fragment；With SEQ ID NO:The homologous protein of 4 fragment 95%；SEQ ID NO:6, with SEQ ID NO:695% homologous protein；SEQ ID NO:6 fragment；And with SEQ ID NO:The homologous protein of 6 fragment 95%. The vaccine can further include the nucleotide sequence of adjuvated protein or coding adjuvated protein.In some embodiments, it is described Adjuvant is IL-12, IL-15, IL-28 or RANTES.

Vaccine comprising nucleic acid molecules can include nucleic acid molecules, and the nucleic acid molecules are comprising selected from group consisting of Nucleotide sequence：SEQ ID NO:1；With SEQ ID NO:195% homologous nucleotide sequence；SEQ ID NO:1 fragment；With SEQ ID NO:The homologous nucleotide sequence of 1 fragment 95%；SEQ ID NO:3；With SEQ ID NO:395% homologous nucleotide sequence； SEQID NO:3 fragment；With SEQ ID NO:The homologous nucleotide sequence of 3 fragment 95%；SEQ ID NO:5；With SEQ ID NO:595% homologous nucleotide sequence；SEQ ID NO:5 fragment；And with SEQ ID NO:The homologous nucleic acid of 5 fragment 95% Sequence.The vaccine can further include the nucleotide sequence of coding adjuvated protein.In some embodiments, the adjuvant is IL-12, IL-15, IL-28 or RANTES.

Brief description

Fig. 1 is the schematic diagram of the structure for illustrating the HBV gene group being made up of the ORF of four overlaps.

Fig. 2A and Fig. 2 B show the result that experiment is expressed from pM Core.Fig. 3 A are shown from In Vitro Translation scheme Result.Fig. 3 B show the result of Western blotting.

Fig. 3 A and Fig. 3 B show the spleen of the C57BL/6 mices that vaccination is carried out come the pM-Core that uses by oneself CD8+ and The amount of the IFN-γ secretion increased in CD4+T cells.

Fig. 4 A and Fig. 4 B show the spleen of the C57BL/6 mices that vaccination is carried out come the pM-Core that uses by oneself CD8+ and The amount of the TNF-α secretion increased in CD4+T cells.

Fig. 5 A and Fig. 5 B show the spleen of the C57BL/6 mices that vaccination is carried out come the pM-Core that uses by oneself CD8+ and The amount of the CD 107a secretions increased in CD4+T cells.

Fig. 6 A and Fig. 6 B show dry in the liver of C57BL/6 mices of vaccination is carried out come the pM-Core that uses by oneself Disturb element-γ t cell responses.

Fig. 7 A and Fig. 7 B show swollen in the liver of C57BL/6 mices of vaccination is carried out come the pM-Core that uses by oneself Tumor necrosis factor-α t cell responses.

Fig. 8 shows the data determined from ELISPOT.

Fig. 9 shows the data from experiment, and the experiment is compared using the cell of CSFE labellings carries out vaccination Do not carry out in the animal of vaccination by the target cell that internal peptide is processed is removed in CD8 T cell bodies.

Figure 10 shows with pVax carriers (control) or with the plasmid pMCore of expression HBV M- cores the CD3+ for processing The comparison of the propagation percentage ratio of CD4+ cells and CD3+CD8+.

Figure 11 A and Figure 11 B are shown come pVax carriers (control) or the plasmid pMCore with expression HBVM- cores of using by oneself The comparison of the anti-HBV core antibodies in the serum being serially diluted of the animal of process.

Figure 12 shows TNF-a the and IFN-g percentage ratios in CD4+ and CD8+ spleens and liver cell.

Figure 13 shows from by measuring the ALT levels in serum whether to determine the removing induced by immune mouse Data in the experiment of impact liver.

Describe in detail

1. define.

Term used herein is not intended to limit merely for the sake of the purpose of description specific embodiment.Such as in explanation Used in book and the claim, in addition to the other clear stipulaties of context, singulative " (a) ", " one kind " and " (the) " is including plural referents (an).

For numerical range cited herein, the number in each insertion having between identical precision is clearly covered Word.For example, the scope for 6-9, is also contemplated by numeral 7 and 8 in addition to 6 and 9, and for the scope of 6.0-7.0, clearly covers Numeral 6.0,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9 and 7.0.

A. adjuvant

" adjuvant " means to be added in DNA plasmid vaccine as herein described to strengthen by hereinafter described as used herein DNA plasmid and nucleic acid sequence encoding coded antigen immunogenic any molecule.

B. antibody

" antibody " means the antibody of type IgG, IgM, IgA, IgD or IgE as used herein, or fragment, its fragment or Derivant, including Fab, F (ab ') 2, Fd and single-chain antibody, double-chain antibody, bi-specific antibody, bifunctional antibody and its spread out It is biological.The antibody can be isolate from the serum sample of mammal antibody, polyclonal antibody, affinity purification antibody Or its mixture, the mixture is to required epi-position or from the enough binding specificities of sequence performance derived from which.

C. coded sequence

" coded sequence " or " code nucleic acid " means the core of the nucleotide sequence comprising coded protein as used herein Sour (RNA or DNA molecular).The coded sequence may further include may be operably coupled to controlling element initial signal and Termination signal, the controlling element include the startup that expression can be instructed in the cell of the individual or mammal of administration of nucleic acid Son and polyadenylation signal.

D. complement

" complement " or " complementation " means that nucleic acid can refer to the nucleotide in nucleotide or nucleic acid molecules as used herein Watson-Crick (Watson-Crick) (for example, A-T/U and C-G) or Hu Sitan (Hoogsteen) alkali between analog Basigamy pair.

E. have or consensus sequence

" have " as used herein or " consensus sequence " means the multiple Asias based on the queue for analyzing specific HBV antigens The peptide sequence of type.The nucleotide sequence of the total peptide sequence of coding can be prepared.Vaccine comprising protein can be used to induction For the extensive immunity of various hypotypes or serotype of specific HBV antigens, the vaccine is had comprising these protein of coding Sequence and/or nucleic acid molecules.

F. electroporation

As " electroporation " used interchangeably herein, " electricity-permeabilization " or " electronic enhancing " (" EP ") mean using across Film electric field pulse is inducing the microcosmic approach (hole) in biomembrane；The presence of which allows biomolecule such as plasmid, few core Thuja acid, siRNA, medicine, ion and water flow to opposite side from the side of cell membrane.

G. fragment

As used herein " fragment " relative to nucleotide sequence mean coding can with total length wild-type strain HBV Cause the nucleotide sequence or one part of the polypeptide of immunoreation in the mammal of antigenic cross-reaction.The fragment can be Selected from least one DNA fragmentation of the various nucleotide sequences of coding protein fragments hereinafter described.

For peptide sequence, " fragment " or " immunogenic fragments " mean can with total length wild-type strain HBV Cause the polypeptide of immunoreation in the mammal of antigenic cross-reaction.The fragment of total albumen can be comprising total albumen extremely Few 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95%.In some embodiments, the fragment for having albumen can include at least 20 aminoacid of total albumen or more, extremely Few 30 aminoacid or more, at least 40 aminoacid or more, at least 50 aminoacid or more, at least 60 aminoacid or More, at least 70 aminoacid or more, at least 80 aminoacid or more, at least 90 aminoacid or it is more, at least 100 Aminoacid or more, at least 110 aminoacid or more, at least 120 aminoacid or more, at least 130 aminoacid or more Many, at least 140 aminoacid or more, at least 150 aminoacid or more, at least 160 aminoacid or it is more, at least 170 Individual aminoacid or more, at least 180 aminoacid or more.

H. gene construct

" gene construct " refers to the DNA or RNA of the nucleotide sequence comprising coded protein as the term is employed herein Molecule.The coded sequence includes the initial signal and termination signal that may be operably coupled to controlling element, the controlling element Including promoter and polyadenylation signal that expression can be instructed in the cell of the individuality of administration of nucleic acid molecule.As herein Term " expression-form " used refers to gene construct, and the gene construct contains and may be operably coupled to coded protein Coded sequence necessary controlling element so that when be present in it is described individuality cell in when, coded sequence will express.

I. it is identical

In the case of two or more nucleic acid or peptide sequence, " identical " or " homogeneity " means as used herein In designated area, sequence has the prescribed percentage of identical residue.The percentage ratio can be calculated by following：Most preferably Compare two sequences, designated area compare two sequences, determine the quantity of the position of identical residue in the two sequences with Produce matched position quantity, with the quantity of matched position divided by the position in the designated area total quantity, and by result It is multiplied by 100 to produce the percentage ratio of sequence iden.There are different length or compare in two sequences and produce one or many In the case that individual end staggeredly and the designated area for comparing only include unique sequence, the residue of unique sequence is included in meter In the denominator of calculation rather than in molecule.When comparison dna and RNA, thymus pyrimidine (T) and uracil (U) are considered With.Homogeneity can be manually or by being performed using computer sequence algorithm such as BLAST or BLAST 2.0.

J. immunoreation

As used herein " immunoreation " mean the introducing in response to the total antigens of antigen such as HBV, the siberian crabapple of host The activation of system (immune system of such as mammal).The immunoreation can be cell effect or humoral response or both Form.

K. nucleic acid

" nucleic acid " or " oligonucleotide " or " polynucleotide " mean at least two for being covalently joined together as used herein Individual nucleotide.Single-stranded description also defines the sequence of complementary strand.Therefore, nucleic acid also covers described single-stranded complementation Chain.Many variants of nucleic acid can be used for and the nucleic acid identical purpose for giving.Therefore, nucleic acid also covers substantially the same Nucleic acid and its complement.The probe that single-stranded offer can be hybridized under strict hybridization conditions with target sequence.Therefore, nucleic acid is also Cover the probe hybridized under stringent hybridization condition.

Nucleic acid can be single-stranded or double-strand or can the part containing both double-strand or single stranded sequence.The core Acid can be both DNA, genome and cDNA, RNA or heterozygote, wherein the nucleic acid can containing deoxyribonucleotide and The combination of ribonucleotide, and it is yellow including uracil, adenine, thymus pyrimidine, cytosine, the fast quinoline of bird, inosine, xanthine time The combination of the base of purine, iso-cytosine and isoguanine.Nucleic acid can pass through chemical synthesis process or pass through restructuring side Method is obtaining.

L. it is operably connected

" it is operably connected " as used herein and means that the expression of gene is the promoter being spatially attached thereto Carry out under control.At the control, promoter can be positioned in 5 ' (upstreams) or 3 ' (downstreams) of gene.The startup Son and the distance between gene can about with the promoter and its base for being controlled in the promoter therefrom gene of derivation The distance between cause is identical.As it is known in the art, the change of this distance can be in the situation for not losing promoter function Under be adjusted.

M. promoter

" promoter " means molecule synthesize or natural source as used herein, and the molecule can give, activate Or the expression of the nucleic acid in enhancing cell.Promoter can include one or more specific transcription regulating nucleotide sequences further to increase Strongly expressed and/or change the expression in its space and/or the expression of time.Promoter can also include Distal enhancer or check unit Part, they may be located at from transcription starting point start it is almost thousand of to base pair at.Promoter can from including virus, Obtain in the source of antibacterial, funguses, plant, insecticide and animal.Promoter can be relative to the cell, group for wherein occurring to express Knit or organ or relative to occur expression stage of development or in response to outside stimuluss such as physiological stress, pathogen, metal ion Or derivant and basically or the distinctively expression of controlling gene component.The representative example of promoter includes that phage t7 starts Son, phage T3 promoter, SP6 promoteres, lac operon-promoter, tac promoteres, SV40 late promoters, SV40 morning Phase promoter, RSV-LTR promoteres, CMV IE promoteres, SV40 early promoters or SV40 late promoters and CMV IE promoteres.

N. signal peptide

" signal peptide " and " targeting sequencing " is used interchangeably herein and refer to and can be connected HBV as herein described The aminoterminal aminoacid sequence of protein.Signal peptide/targeting sequencing is indicated generally at the position of protein.Letter used herein Number peptide/targeting sequencing preferably facilitates protein and secretes from producing in its cell.Signal peptide/targeting sequencing is usually from protein Remainder cracking, protein Jing after secreting from cell is commonly referred to as mature protein.Signal peptide/targeting sequencing connects It is connected on the N-terminal of the protein.N-terminal with regard to signal peptide or targeting sequencing to be connected to protein as mentioned in this article, letter Number peptide/targeting sequencing replaces the methionine of the N-terminal of the protein encoded by the start codon of nucleotide sequence, rather than is not having Code for said proteins in the case of signal coding sequence.Therefore for example, SEQ ID NO:4 is to carry to be connected to SEQ ID NO:The SEQ ID NO of 2 N-terminal signal peptide/targeting sequencing:2, i.e. SEQ ID NO:4 is to include to be connected to SEQ ID NO:2 N The protein of the signal peptide at end.In SEQ ID NO:The first residue " Xaa " in 2 is typically methionine when there is no signal peptide. However, include being connected to SEQ ID NO:The protein of 2 signal peptide such as SEQ ID NO:4 being connected to the egg by signal peptide The residue of white matter replaces 1 methionine of residue at Xaa.Therefore, SEQ ID NO:2 N-terminal residue can be any aminoacid, But it is methionine if it is by homing sequence coding.Signal peptide/targeting sequencing is connected to SEQ ID NO:2 N-terminal Generally eliminate N-terminal methionine.As used herein, it is desirable to SEQ ID NO:4 comprising with being connected to SEQ ID NO:2 N The SEQ ID NO of the signal peptide/targeting sequencing at end:2, although eliminating SEQ ID NO:2 N section Xaa residues.Similarly, SEQ ID NO:4 coded sequence includes SEQ ID NO:2 coded sequence encodes SEQ ID NO with being connected to:2 code sequence The coded sequence of the 5 ' signal peptide/targeting sequencings held of row.In SEQ ID NO:In 2 coded sequence, start codon can be " nnn ", but when the coded sequence of signal peptide/targeting sequencing is connected to coding SEQ ID NO:During 5 ' end of 2 coded sequence, Which is eliminated.As used herein, it is desirable to SEQ ID NO:4 coded sequence includes SEQ ID NO:2 coded sequence and company It is connected on the SEQ ID NO for nnn occur:The coded sequence of signal peptide/targeting sequencing that the 5 ' of 2 coded sequence are held.Therefore, example Such as, it is desirable to SEQ ID NO:3 include SEQ ID NO:1 and it is connected to SEQ ID NO:Signal peptide/targeting sequencing that the 5 ' of 1 are held Coded sequence, replaces nnn.In some embodiments, nnn is in SEQ ID NO:The start codon that the 5 ' of 1 are held.

O. strict hybridization conditions

" strict hybridization conditions " mean such condition as used herein, i.e. such as answering in nucleic acid under the described conditions In hybrid compound, the first nucleotide sequence (for example, probe) will be hybridized with second nucleotide sequence (for example, target).Strict bar Part is sequence dependent and will be different in different environment.Strict condition can under ionic strength pH for limiting To be selected as the thermal melting point (T than particular sequence_m) low about 5-10 DEG C.The T_mCan be that such temperature (is being limited Under ionic strength, pH and nucleic acid concentration), at said temperatures with the 50% of target-complementary probe and target sequence in equilibrium-like Hybridized under state (because target sequence is present in excess, in T_mUnder, 50% probe is occupied in the state of the equilibrium).Stringent condition Can be those conditions, i.e., wherein salinity is less than about the sodium ion of 1.0M, such as the about 0.01-1.0M under pH 7.0 to 8.3 Na ion concentration (or other salt), and temperature for short probe (for example, about 10-50 nucleotide) at least about 30 DEG C and For long probe (for example, greater than about 50 nucleotide) is at least about 60 DEG C.Strict condition can also be gone steady by addition Determine agent such as Methanamide to realize.For hybridization select or specific, positive signal can be at least the 2 to 10 of background hybridization Times.Exemplary strict hybridization conditions include following：50% Methanamide, 5x SSC and 1%SDS, the incubation at 42 DEG C, or 5x SSC, 1%SDS, at 65 DEG C incubation, washed with 0.2x SSC and 0.1%SDS at 65 DEG C.

P. it is substantially complementary

" be substantially complementary " as used herein mean First ray 8,9,10,11,12,13,14,15,16,17,18, 19、20、21、22、23、24、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、180、270、 360th, 450,540 or more nucleotide or aminoacid region in the complement at least 60% of the second sequence, 65%, 70%, 75%th, 80%, 85%, 90%, 95%, 97%, 98% or 99% are identical, or to mean that two sequences are carried out under strict hybridization conditions miscellaneous Hand over.

Q. it is substantially the same

As used herein " substantially the same " mean First ray and the second sequence 8,9,10,11,12,13,14, 15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、 100th, 180,270,360,450,540 or more nucleotide or amino acid region in be at least 60%, 65%, 70%, 75%, 80%, 85%th, 90%, 95%, 97%, 98% or 99% identical, or for nucleic acid, if the complement of First ray and the second sequence It is substantially complementary, then First ray and the second sequence are also so identical.

R. hypotype or serotype

" hypotype " or " serotype "：As herein exchange use and with regard to HBV, it is intended that the genetic mutation of HBV so that One hypotype is recognized and separated from different hypotypes by immune system.

S. variant

" variant " used for nucleic acid means a part or fragment for (i) reference nucleotide sequence herein；(ii) join The complement of examination nucleotide sequence or part thereof；(iii) nucleic acid substantially the same with reference nucleic acid or its complement；Or (iv) nucleic acid under strict conditions with reference nucleic acid, its complement or the sequence hybridization substantially the same with which.

" variant " for peptide or polypeptide is by the insertion of aminoacid, disappearance or conservative replaces in aminoacid sequence Upper difference, but retain at least one biological activity.Variant is still meant that with the aminoacid substantially the same with reference protein The protein of sequence, the reference protein have the aminoacid sequence for retaining at least one biological activity.Aminoacid it is conservative Property replace, i.e., replace amino with the different aminoacids of similar characteristic (for example, hydrophilic, the degree of charging zone and distribution) Acid, is considered as being usually directed to minor variations in the art.As understood in the art, these minor variations partly can pass through Consider the hydrophilic and hydrophobic index of aminoacid recognizing.Kate (Kyte) etc., J. Mol. BioL (J.Mol.Biol.) 157: 105-132(1982).The hydrophilic and hydrophobic index of the aminoacid is the consideration based on its hydrophobicity and electric charge.It is known in the art It is that the aminoacid of similar hydrophilic and hydrophobic index can be substituted and still retaining protein function.In one aspect, hydrophobe Sex index is substituted for ± 2 aminoacid.The hydrophilic of aminoacid can be utilized to disclose and can produce reservation biological function The replacement of protein.Consider that the hydrophilic of aminoacid allows the local average for calculating the peptide maximum hydrophilic in the case of peptide Property, this is the useful measurement well associated with antigenicity and immunogenicity that has been reported.U.S. Patent number 4,554,101 with It is fully incorporated by reference into herein.As this area understands, the replacement of the aminoacid with similar hydrophilicity score can be produced Retain the peptide of biological activity (such as immunogenicity).The available aminoacid with hydrophilicity value each other in ± 2 is replaced. Both the hydrophilic and hydrophobic index of aminoacid and hydrophilicity value is affected by the specific side chain of the aminoacid.It is consistent with the observation , the aminoacid replacement compatible with biological function be understood to the similarity relative depending on these aminoacid, and especially It is the side chain of those aminoacid, as disclosed in by hydrophobicity, hydrophilic, electric charge, size and other characteristics.

T. carrier

" carrier " means the nucleotide sequence containing replication orgin as used herein.Carrier can be viral vector, phagocytosis Body, bacterial artificial chromosome or yeast artificial chromosome.Carrier can be DNA or RNA carriers.Carrier can be self replication Dye external carrier, and preferably DNA plasmid.

2.HBV cAgs

HbcAg represent by induction 1) cytotoxic T lymphocyte (CTL) reaction, 2) t helper cell reaction with And/or 3) B cell reaction or preferably above-mentioned all of immune-mediated virus sweep important target, come for intersect be in Pass.

Table 2 is shown from HBV-A, HBV-B, HBV-C, HBV-D and HBV-E genotype with total HBV cores egg Similarity between the genotype of white cAg, the cAg are referred to as " HBV-M- cores " in the graph.For Some embodiments, HBV M core constructs are configured to have the homology of the increase to extensive HBV cores target.Tool Have designed M- core constructs cAg genotype between similarity-increased to extensive HBV cores target Homology.All of genotype should be represented with the general immunisation treatment vaccine of HBV.

Table 2

Provided herein is the antigen of the immunoreation for one or more HBV serotype can be caused in mammal. The antigen can be induced for the immunogen comprising making them particularly effectively as immunogenic core protein epi-position Anti- HBV immunoreation.HBV antigens can include total length translation product, its variant, its fragment or its combination.

There is provided total HBcAg (SEQ ID NO:2).Generation is included in the N-terminal of HBcAg consensus sequence The aminoacid sequence of IgE targeting sequencings.Therefore, additionally provide to have and be connected to total HBcAg (SEQ ID NO:2) IgE targeting sequencings (SEQ ID NO:7) protein, to provide IgE targeting sequencings-total HBcAg (SEQ ID NO: 4).Some embodiments for providing also include HA labellings (the SEQ ID NO of the C-terminal for being connected to HBcAg consensus sequence: 8).It thus provides total albumen (the SEQ ID NO of HBcAg:6), the total albumen is included and is connected to HBV cores Albumen consensus sequence (SEQ ID NO:2) IgE targeting sequencings (SEQ ID NO:7) and it is connected to the total sequence of HBcAg HA labellings (the SEQ ID NO of the C-terminal of row:8).

Protein can be with SEQ ID NO:2、SEQ ID NO:4 and SEQ ID NO:6 is homologous.Some embodiments are related to And immunogenic protein of the total protein sequence with 95% homology with this paper.Some embodiments are related to this paper's Total protein sequence has the immunogenic protein of 96% homology.Some embodiments are related to the total albumen sequence with this paper Immunogenic protein of the row with 97% homology.Some embodiments are related to same with 98% with the total protein sequence of this paper The immunogenic protein of source property.Some embodiments are related to the immunity for having 99% homology with the total protein sequence of this paper Genic protein.

In some embodiments, the protein does not have targeting sequencing.In some embodiments, the protein does not have There are IgE targeting sequencings.The fragment of total albumen can include total albumen at least 10%, at least 15%, at least 20%, at least 25%th, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.Can To provide SEQ ID NO:2、SEQ ID NO:4 and SEQ ID NO:6 immunogenic fragments.Immunogenic fragments can be wrapped The NO of ID containing SEQ:2、SEQ ID NO:4 and SEQ ID NO:6 at least 10%, at least 15%, at least 20%, at least 25%, at least 30%th, at least 35%, at least 40%, at least 45%, at least 50% or at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, At least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.In some enforcements In scheme, fragment includes targeting sequencing, such as such as immunoglobulin leader sequence, IgE targeting sequencings.In some embodiments In, fragment does not have targeting sequencing.In some embodiments, fragment does not have targeting sequencing, i.e. IgE targeting sequencings.

Can provide with SEQ ID NO:2、SEQ ID NO:4 and SEQ ID NO:6 immunogenic fragments are same The immunogenic fragments of the protein of the aminoacid sequence in source.Such immunogenic fragments can comprising with S SEQ ID NO:2、 SEQ ID NO:4 and SEQ ID NO:695% homologous protein at least 10%, at least 15%, at least 20%, at least 25%, at least 30%th, at least 35%, at least 40%, at least 45%, at least 50% or at least 55% at least 60%, at least 65%, at least 70%, at least 75%, At least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.Some embodiment party Case is related to the immunogenic fragments for having 96% homology with the immunogenic fragments of the total protein sequence of this paper.Some enforcements Scheme is related to the immunogenic fragments for having 97% homology with the immunogenic fragments of the total protein sequence of this paper.Some realities The scheme of applying is related to the immunogenic fragments for having 98% homology with the immunogenic fragments of the total protein sequence of this paper.Some Embodiment is related to the immunogenic fragments for having 99% homology with the immunogenic fragments of the total protein sequence of this paper. In some embodiments, fragment includes targeting sequencing, such as such as immunoglobulin leader sequence, IgE targeting sequencings.In some realities Apply in scheme, fragment does not have targeting sequencing.In some embodiments, fragment does not have targeting sequencing, i.e. IgE targeting sequencings.

3. gene order, construct and plasmid

Coding SEQ ID NO can routinely be produced:2、SEQ ID NO:4 and SEQ ID NO:6 and homologous protein, The nucleotide sequence of the immunogenic fragments of immunogenic fragments and homologous protein.It is, therefore, possible to provide coding is orderly together Row are up to 96% homology with up to 95% homology and consensus sequence and consensus sequence is up to 96% homology, orderly together Row are up to 97% homology and consensus sequence is reached high 98% homology and the immunogenicity of 99% homology is up to consensus sequence The nucleic acid molecules of protein.Similarly, additionally provide coding immunogenic fragments as herein described and with albumen as herein described The nucleotide sequence of the immunogenic fragments of the homologous protein of matter.

Produce the nucleic acid molecules of coding consensus amino acid sequences.Vaccine can include one or more total form of coding One or more nucleotide sequence of immunogenic protein, the immunogenic protein are steady in people to optimize selected from producing This group of the sequence of qualitative and expression.Total albumen (the SEQ ID NO of coding HBcAg:2) nucleotide sequence (SEQ ID NO:1) total albumen (the SEQ ID NO of IgE targeting sequencings-HBcAg, are encoded:4) nucleotide sequence (SEQ ID NO:And total albumen-HA labellings (the SEQ ID NO of coding IgE targeting sequencings-HBcAg 3):6) nucleotide sequence (SEQ ID NO:5).Some embodiments are related to the same with 95% with this paper nucleic acid coding sequences of encoding immunogenic proteins The nucleic acid molecules of source property.What some embodiments were related to encoding immunogenic proteins has 96% with this paper nucleic acid coding sequences The nucleic acid molecules of homology.Some embodiments are related to having with this paper nucleic acid coding sequences for encoding immunogenic proteins The nucleic acid molecules of 97% homology.Some embodiments are related to having with this paper nucleic acid coding sequences for encoding immunogenic proteins There are the nucleic acid molecules of 98% homology.Some embodiments be related to encoding immunogenic proteins with this paper nucleic acid coding sequences Nucleic acid molecules with 99% homology.In some embodiments, with the coded sequence with total albumen disclosed herein The nucleic acid molecules of homologous coded sequence disclosed herein being connected to comprising coding IgE targeting sequencings encodes disclosed herein Homologous protein sequence coded sequence the 5 ' sequences held.

In some embodiments, the nucleotide sequence does not have the coded sequence of encoding leader sequence.In some embodiment party In case, the nucleotide sequence does not encode the coded sequence of IgE targeting sequencings.

Some embodiments are related to SEQ ID NO:1、SEQ ID NO:3 and SEQ ID NO:5 fragment.Fragment can be SEQ ID NO:1、SEQ ID NO:3 and SEQ ID NO:5 at least 10%, at least 15%, at least 20%, at least 25%, at least 30%th, at least 35%, at least 40%, at least 45%, at least 50% or at least 55% at least 60%, at least 65%, at least 70%, at least 75%, At least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%.Fragment can be with SEQ ID NO:1、SEQ ID NO:3 and SEQ ID NO:5 fragment at least 95%, at least 96%, at least 97%, at least 98% or At least 99% is homologous.In some embodiments, sequence of the fragment comprising encoding leader sequence, such as immunoglobulin leader sequence Row, such as IgE targeting sequencings.In some embodiments, fragment does not have the coded sequence of encoding leader sequence.In some embodiment party In case, fragment does not have encoding leader sequence, the i.e. coded sequence of IgE targeting sequencings.

Provided herein is the gene construct of the nucleotide sequence of coding HbcAg disclosed herein, institute can be included State cAg include the homologous sequence of total protein sequence and total protein sequence, the fragment of total protein sequence and with The homologous sequence of the fragment of total protein sequence.The gene construct can as functional genomics outside molecule and deposit .The gene construct can be the linear mini-chromosome for including centromere, telomere or plasmid or cosmid.

The gene construct can also be a part for the genome of recombinant viral vector, the recombinant viral vector bag Include recombinant adenoviruss, recombinant adeno-associated virus and recombinant vaccinia.Gene construct can be the viable microbial in attenuation Or the part of the hereditary material in the recombinant microorganism carrier in cell living.

Gene construct can include the controlling element of the gene expression of the coded sequence for nucleic acid.Controlling element can be with It is promoter, enhancer, start codon, termination codon or polyadenylation signal.

Nucleotide sequence can constitute can be carrier gene construct.The carrier can be effectively drawing in mammal Send out immunoreation amount in the cell of mammal antigen expressed.Institute+state carrier can be recombinant.The carrier can be wrapped Heterologous nucleic acids containing coding for antigens.The carrier can be plasmid.The carrier go for the nucleic acid of coding for antigens come Transfectional cell, the host cell of the conversion are cultivated and are maintained under conditions of there is antigen presentation wherein.

Coded sequence can be optimized to be used in the stability and high level of expression.In some cases, select codon To reduce the formation of RNA secondary structures, the secondary structure for such as being formed due to intramolecular bond.

The carrier can include the heterologous nucleic acids of coding for antigens, and can further comprising can be in antigen encoding sequence The start codon of the upstream of row and can be in the termination codon in the downstream of antigen encoding sequences.Start codon and termination are close Numeral can be with antigen encoding sequences in frame.The carrier is also comprising the startup that may be operably coupled to antigen encoding sequences Son.The promoter that may be operably coupled to antigen encoding sequences can be from the promoter of simian virus 40 (SV40), mice The long end weight of mammary tumour virus (MMTV) promoter, human immunodeficiency virus (HIV) promoter such as bovine immunodeficiency viruss (BIV) Multiple (LTR) promoter, Moloney (Moloney) viral promotors, avian leukosis virus (ALV) promoter, cytomegaloviruses (CMV) promoter such as CMV immediate early promoters, love bar Er Shi (Epstein Barr) virus (EBV) promoter or Lloyd's's meat Tumor virus (RSV) promoter.The promoter can also be the promoter from people's gene, and the people's gene such as people's flesh moves egg In vain, people's myosin, people's haemachrome, people's muscle creatin or human metal thioalbumen.The promoter can also be tissue specificity Promoter, such as naturally occurring or synthetic muscle or skin-specific promoter.The example of these promoteres is public in U.S. Patent application It is described in cloth US20040175727, the entire content of the Shen Qing Publication is incorporated herein.

The carrier can also include polyadenylation signal, and the polyadenylation signal can be in HBV cores The downstream of albumen coded sequence.The polyadenylation signal can be SV40 polyadenylation signals, LTR polyadenosines Polyadenylation signal, bovine growth hormone (bGH) polyadenylation signal, human growth hormone's (hGH) polyadenylation signal or people Betaglobulin polyadenylation signal.The SV40 polyadenylation signals can be from pCEP4 carriers (hero company (Invitrogen), Santiago (San Diego), California (CA)) polyadenylation signal.

The carrier is further included in the enhancer of total HBcAg upstream of coding sequence.The enhancer pair It is necessary to express in DNA.The enhancer can be human actin, people's myosin, people's haemachrome, people's muscle creatin or Virus enhancer, such as from a kind of enhancer of CMV, HA, RSV or EBV.Polymerized nucleoside acid function enhancer is described in U.S. In state's patent No. 5,593,972,5,962,428 and WO94/016737, the full content of each patent is incorporated by reference Herein.

Carrier can also include the replication orgin of mammal, so that carrier is maintained dyeing in vitro and the product in cell Multiple copies of raw carrier.The carrier can be from hero company (Santiago, California) pVAX1, PCEP4 or pREP4, which can include the replication orgin and nuclear antigen EBNA-1 coding region of love bar Er Shi virus, and this can be with High copy episomal replication is produced in the case of without integrating.The carrier can be the pVAX1 with change or pVax1 Variant, variant plasmid as described herein.The variant pVax1 plasmids are Backbone Vector plasmid pVAX1 (hero company, karrs This Ahmedabad (Carlsbad), California) 2998 base pair variants.The CMV promoter is located at base 137-724 Place.T7 promoteres/priming site is located at base 664-683.Multiple clone site is located at base 696-811.Cattle GH poly glands Nucleotide signal is at base 829-1053.Kanamycin (Kanamycin) resistant gene is at base 1226-2020. PUC starting points are at base 2320-2993.

Based on the sequence of the pVAX1 that can be obtained from hero company, in the main chain for being used as plasmid 1-6 as herein described Following mutation is found in the sequence of pVAX1：

The main chain of the carrier can be pAV0242.The carrier can be 5 type adenoviruss (Ad5) carrier of replication defective.

The carrier can also include regulating and controlling sequence, and the regulating and controlling sequence can be highly suitable for the food in one's mouth for applying the carrier Gene expression in newborn animal or people's cell.Total HBV coded sequences can include codon, and the codon can be allowed Coded sequence is transcribed in host cell more effectively.

The carrier can be pSE420 (hero company, Santiago, California (Calif.)), and which can be used for Escherichia coli (Escherichia coli) (E.coli) in produce protein.The carrier can be that (hero is public for pYES2 Department, Santiago, California), which can be used for the Wine brewing yeast strain (Saccharomyces in yeast Cerevisiae strains) middle generation protein.The carrier can also have MAXBAC^TMComplete baculovirus expression system System (hero company, Santiago, California), which can be used to protein is produced in insect cell.The carrier is also Can be pcDNA I or pcDNA3 (hero company, Santiago, California), which can be used for thin in mammal Protein is produced in born of the same parents such as Chinese hamster ovary (CHO) cell.The carrier can be available with easy by routine techniquess Producing protein expression carrier or system, the technology and material include Pehanorm Brooker (Sambrook) to initial substance Deng, molecular cloning and laboratory manual (Molecular Cloning and Laboratory Manual), the second edition, cold spring Lane laboratory (Cold Spring Harbor) (1989), which is fully incorporated herein by reference.

4. pharmaceutical composition

Provided herein is pharmaceutical composition of the invention, DNA of the compositionss comprising about 1 nanogram to about 10mg. In some embodiments, pharmaceutical composition of the invention be included in it is following between：1) at least 10,15,20,25,30,35, 40th, 45,50,55,60,65,70,75,80,85,90,95 or 100 nanograms or at least 1,5,10,15,20,25,30,35,40, 45、50、55、60、65、70、75、80、85、90、95、100、105、110、115、120、125、130、135、140、145、150、 155、160、165、170、175、180、185、190、195、200、205、210、215、220、225、230、235、240、245、 250、255、260、265、270、275、280、285、290、295、300、305、310、315、320、325、330、335、340、 345、350、355、360、365、370、375、380、385、390、395、400、405、410、415、420、425、430、435、 440、445、450、455、460、465、470、475、480、485、490、495、500、605、610、615、620、625、630、 635、640、645、650、655、660、665、670、675、680、685、690、695、700、705、710、715、720、725、 730、735、740、745、750、755、760、765、770、775、780、785、790、795、800、805、810、815、820、 825、830、835、840、845、850、855、860、865、870、875、880、885、890、895.900、905、910、915、 920th, 925,930,935,940,945,950,955,960,965,970,975,980,985,990,995 or 1000 micrograms or extremely Few 1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,7.5,8,8.5,9,9.5 or 10mg or more；And 2) up to And including 15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100 nanograms, or up to and wrap Include 1,5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,105,110,115, 120、125、130、135、140、145、150、155、160、165、170、175、180、185、190、195、200、205、210、 215、220、225、230、235、240、245、250、255、260、265、270、275、280、285、290、295、300、305、 310、315、320、325、330、335、340、345、350、355、360、365、370、375、380、385、390、395、400、 405、410、415、420、425、430、435、440、445、450、455、460、465、470、475、480、485、490、495、 500、605、610、615、620、625、630、635、640、645、650、655、660、665、670、675、680、685、690、 695、700、705、710、715、720、725、730、735、740、745、750、755、760、765、770、775、780、785、 790、795、800、805、810、815、820、825、830、835、840、845、850、855、860、865、870、875、880、 885、890、895.900、905、910、915、920、925、930、935、940、945、950、955、960、965、970、975、 980th, 985,990,995 or 1000 microgram, or up to and including 1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7, 7.5th, 8,8.5,9,9.5 or 10mg.In some embodiments, pharmaceutical composition of the invention includes about 5 nanograms to about The DNA of 10mg.In some embodiments, DNA of the pharmaceutical composition of the invention comprising about 25 nanograms to about 5 milligrams. In some embodiments, described pharmaceutical composition contains the DNA. of about 50 nanograms to about 1 milligram in some embodiments, institute State the DNA that pharmaceutical composition contains about 0.1 microgram to about 500 micrograms.In some embodiments, described pharmaceutical composition contains The DNA of about 1 microgram to about 350 micrograms.In some embodiments, described pharmaceutical composition contains about 5 micrograms to about 250 micrograms DNA.In some embodiments, described pharmaceutical composition contains the DNA of about 10 micrograms to about 200 micrograms.In some enforcements In scheme, described pharmaceutical composition contains the DNA of about 15 micrograms to about 150 micrograms.In some embodiments, the medicine group Compound contains the DNA of about 20 micrograms to about 100 micrograms.In some embodiments, described pharmaceutical composition contains about 25 micrograms To the DNA of about 75 micrograms.In some embodiments, described pharmaceutical composition contains the DNA of about 30 micrograms to about 50 micrograms. In some embodiments, described pharmaceutical composition contains the DNA of about 35 micrograms to about 40 micrograms.In some embodiments, institute State the DNA that pharmaceutical composition contains about 100 micrograms to about 200 micrograms.In some embodiments, described pharmaceutical composition is included The DNA of about 10 micrograms to about 100 micrograms.In some embodiments, described pharmaceutical composition is micro- to about 80 comprising about 20 micrograms Gram DNA.In some embodiments, DNA of the described pharmaceutical composition comprising about 25 micrograms to about 60 micrograms.In some enforcements In scheme, DNA of the described pharmaceutical composition comprising about 30 nanograms to about 50 micrograms.In some embodiments, the medicine group DNA of the compound comprising about 35 nanograms to about 45 micrograms.In some preferred embodiments, described pharmaceutical composition contains about The DNA of 0.1 microgram to about 500 micrograms.In some preferred embodiments, described pharmaceutical composition contains about 1 microgram to about The DNA of 350 micrograms.In some preferred embodiments, described pharmaceutical composition contains about 25 micrograms to about 250 micrograms DNA.In some preferred embodiments, described pharmaceutical composition contains the DNA of about 100 micrograms to about 200 micrograms.

Pharmaceutical composition of the invention is prepared according to method of application to be used.It is injectable in pharmaceutical composition Pharmaceutical composition in the case of, they are aseptic, apyrogeneitys and agranular.Preferably use isotonic preparation.Generally, it is isotonic Additive may include Sodium Chloride, glucose, Mannitol, Sorbitol and Lactose.In some cases it may be preferred to isosmotic solution such as phosphorus Hydrochlorate buffer saline.Stabilizer includes gelatin and albumin.In some embodiments, vasoconstrictor is added to into preparation In.

Preferably, described pharmaceutical composition is vaccine, and more preferably DNA vaccination.

Provided herein is the epidemic disease of the immunoreation of one or more genotype for HBV can be produced in mammal Seedling.The vaccine may include gene construct as discussed above.

Although being not affected by the constraint of scientific theory, vaccine can be used to cause one or more gene extensively for HBV The immunoreation (humoral immunization, cellular immunization or the two) of type.Vaccine can be comprising total HBcAg sequence (SEQ ID NO:2) coded sequence；It is connected to total HBcAg sequence (SEQ ID NO:4) IgE targeting sequencings；And connection To the IgE targeting sequencings of total HBcAg, the core protein is connected to HA labelled sequence (SEQ ID NO:6).Vaccine Can be comprising total HBcAg sequence (SEQ ID NO:2) special coded sequence such as (SEQ ID NO:1)；It is connected to altogether There are HBcAg sequence (SEQ ID NO:4) IgE targeting sequencings such as (SEQ ID NO:3)；And it is connected to total HBV The IgE targeting sequencings of core protein such as (SEQ ID NO:5), the core protein is connected to HA labelled sequence (SEQ ID NO: 6)。

Some alternate embodiments include those comprising the following nucleotide sequence of coding：Total HBcAg is exempted from One or more homologous protein of epidemic disease immunogenic fragment and total HBcAg and homologous with total HBcAg The immunogenic fragments of one or more protein.

Some embodiments provide the method for producing the immunoreation for HBcAg, and methods described is included to individual Body applies one or more compositions as herein described.Some embodiments provide prophylactically to enter individuality for HBV infection The method of row vaccination, methods described include applying one or more compositions as herein described.Some embodiments are provided The method that therapeutic ground carries out vaccination to the individuality that infects HBV, methods described include applying it is as herein described a kind of or Numerous compositions.The diagnosis of HBV infection can be routinely completed before administration.

The vaccine can be DNA vaccination.The DNA vaccination can include various identical or different plasmids, the matter Nucleotide sequence of the grain comprising the total HBcAg of coding.

DNA vaccination is disclosed in U.S. Patent number 5,593,972,5,739,118,5,817,637,5,830,876,5,962, 428th, 5,981,505,5,580,859,5,703,055 and 5, in 676,594, the United States Patent (USP) is by reference all simultaneously Enter herein.The DNA vaccination can further comprising prevention, which be incorporated into the element in chromosome or reagent.The vaccine can Being the RNA of HBcAg.The RNA vaccines are directed in cell.

The vaccine can be the recombiant vaccine comprising gene construct mentioned above or antigen.The vaccine can be with Have HBcAg, be total to comprising one or more comprising one or more in one or more protein subunit forms There are one or more killed virion of HBcAg or subtracting comprising one or more total HBcAg Malicious virion.The vaccine of the attenuation can be the live vaccine of attenuation, killed vaccine or using recombinant vector delivering The vaccine and subunit vaccine of exogenous gene and glucoprotein vaccine, the exogenous gene encode one or more total HBV core Heart protein.Attenuated live vaccine, using recombinant vector delivering those vaccines, subunit vaccine and the glucoprotein vaccine of exogenous antigen Example be described in U.S. Patent number：4,510,245；4,797,368；4,722,848；4,790,987；4,920,209；5, 017,487；5,077,044；5,110,587；5,112,749；5,174,993；5,223,424；5,225,336；5,240, 703；5,242,829；5,294,441；5,294,548；5,310,668；5,387,744；5,389,368；5,424,065；5, 451,499；5,453,364；5,462,734；5,470,734；5,474,935；5,482,713；5,591,439；5,643, 579；5,650,309；5,698,202；5,955,088；6,034,298；6,042,836；6,156,319 and 6, in 589,529, Each of which is herein incorporated by reference.

The vaccine can comprising the carrier for the multiple HBV gene types from multiple given areas in the world and/or Protein.The vaccine for being provided can be used to induce immunoreation, the immunoreation to include that the property controlled is treated or preventative immunity is anti- Should.Can produce for total HBcAg the and also extensive antibody for crossing over the viral Multi-genotypes of HBV and/or kill Hinder T cell.This antibody-like and cell can be separated.

The vaccine can further include pharmaceutically acceptable excipient.The pharmaceutically acceptable excipient can To be the functional molecular as vehicle, adjuvant, carrier (carrier) or diluent.The pharmaceutically acceptable excipient Can be transfection, which may include that surfactant such as immunostimulating complex (ISCOMS), Fei Shi (Freunds) be not complete Full adjuvant, LPS analog (including single phosphoramide A), muramyl peptide, benzoquinone analog, vesicle such as Squalene and Squalene, hyalomitome Acid, lipid, liposome, calcium ion, virus protein, polyanion, polycation or nanoparticle or other known transfection Accelerator.

The transfection is polyanion, polycation (including Poly-L-glutamic acid (LGS)) or lipid.Described turn Dye accelerator is Poly-L-glutamic acid, and it is highly preferred that the Poly-L-glutamic acid is present in epidemic disease with the concentration less than 6mg/ml Seedling.The transfection can also include surfactant such as immunostimulating complex (ISCOMS), Fei Shi Freund's incomplete adjuvants, LPS analog (including single phosphoramide A), muramyl peptide, benzoquinone analog and vesicle such as Squalene and Squalene, and hyaluronic acid Can also be used to apply together with the gene construct.In some embodiments, the DNA vector vaccine can also include Transfection such as lipid, liposome (include lecithin liposome or other liposomees known in the art (such as DNA- liposomees Mixture (see, for example, W09324640))), calcium ion, virus protein, polyanion, polycation or nanoparticle or its Transfection known to it.Preferably, the transfection is polyanion, polycation (including Poly-L-glutamic acid ) or lipid (LGS).Concentration of the transfection agents in the vaccine be less than 4mg/ml, less than 2mg/ml, less than 1mg/ml, be less than 0.750mg/ml, less than 0.500mg/ml, less than 0.250mg/ml, less than 0.100mg/ml, less than 0.050mg/ml or be less than 0.010mg/ml。

The pharmaceutically acceptable excipient can be adjuvant.The adjuvant can be substitute plasmid in expression or Other genes for being delivered as protein and the above plasmid combinations in vaccine.The adjuvant can be selected from the following group Into group：Alpha-interferon (IFN-α), beta-interferon (IFN-β), gamma interferon, platelet derived growth factor (PDGF), TNF α, TNF β, GM-CSF, epidermal growth factor (EGF), the T cell of skin attract chemotactic factor (CTACK), the expression of epithelium thymus to become Changing the relevant epithelium chemotactic factor (MEC) of the factor (TECK), mucosa, IL-12, IL-15, MHC, CD80, CD86 (includes disappearance letter Number sequence simultaneously optionally includes the IL-15 of signal peptide from IgE).The adjuvant can be IL-12, IL-15, IL-28, CTACK, TECK, platelet derived growth factor (PDGF), TNF α, TNF β, GM-CSF, epidermal growth factor (EGF), IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18 or its combination.

Can be that other genes of applicable adjuvant include encoding those following genes：MCP-1、MIP-1a、MIP-1p、 IL-8, RANTES, L-selectin, palatelet-selectin, E-Selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, IL-18's is prominent Deformation type, CD40, CD40L, angiogenesis factor, fibroblast growth factor, IL-7, nerve growth factor, blood vessel endothelium Somatomedin, Fas, TNF receptor, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, P65Re1, MyD88, IRAK, TRAF6, IkB, inactivation NIK, SAP K, SAP-1, JNK, ifn response gene, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK part, Ox40, Ox40 part, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1, TAP2 and its function fragment.

5. the method for delivering

Provided herein is for the method for delivering pharmaceutical preparation, preferred vaccine, for providing the HBV core eggs comprising epi-position White gene construct and protein, the epi-position make them become particularly effective immunogen, can lure for the immunogen Lead the immunoreation to HBV virus infection.The method for delivering the vaccine or vaccination can be provided to inducing therapeutic And/or preventative immunoreation.The vaccination can produce exempting from for various HBV gene types in mammal Epidemic disease is reacted.The vaccine may be delivered into the individual immune activity to adjust mammal and strengthen immune anti- Should.The delivering of the vaccine can be the transfection of the HA antigens as nucleic acid molecules, and the nucleic acid molecules are expressed in cell simultaneously The surface of cell is delivered to, immune system identification inducing cellular immune, humoral immunization or cell and body on cell surface Liquid immunity.The delivering of vaccine can be used to induce in mammal by applying vaccine as discussed herein to mammal Or cause for various HBV viral immunoreation.

The mammal is being given and therefore by the cell of the vehicle delivery to mammal by the vaccine delivery Afterwards, the cell of transfection will be expressed and be secreted total HBcAg.The protein or synthetic antigen of these secretions will be used as external source Material and by immune system recognize, this will carry out including following immunoreation：Generate for the antigen antibody and T cell responses of the specificity for the antigen.In some embodiments, moved with the suckling of vaccination discussed in this article Thing is by the immune system with initiation, and when being excited by HBV Strain, the immune system of initiation would allow through body fluid and exempt from Epidemic disease, cellular immunization or both are viral quickly to remove subsequent HBV.The vaccine may be delivered into individuality to adjust individuality Immune activity, so as to strengthen immunoreation.

The vaccine can be delivered in the form of DNA vaccination and the method for DNA delivery vaccine is described in U.S. Patent number 4,945,050 and 5,036,006, both it is fully incorporated herein by reference.

The vaccine can be applied to mammal to cause immunoreation in mammal.The mammal can be with Be people, non-human primates, cow, pig, sheep, goat, Saigae Tataricae, wild ox, Babalus bubalis L., bovid, deer, Rrinaceus earopaeuss, as, camel, sheep Camel, mice, rat or chicken, and be preferably people, cow, pig or chicken.

A. combined therapy

Described pharmaceutical composition, preferably vaccine as herein described can be applied with the assortment of genes of protein or coding adjuvant With the adjuvant can include：Alpha-interferon (IFN-α), beta-interferon (IFN-β), gamma interferon, IL-12, IL-15, IL- 28th, CTACK, TECK, platelet derived growth factor (PDGF), TNF α, TNF β, GM-CSF, epidermal growth factor (EGF), IL- 1st, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L- choosing Select element, palatelet-selectin, E-Selectin, CD34, GlyCAM-1, MadCAM-1, LFA-1, VLA-1, Mac-1, pl50.95, The mutant form of PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, IL-18, CD40, CD40L, angiogenesis factor, fibroblast growth factor, IL-7, nerve growth factor, VEGF, Fas, TNF receptors, Flt, Apo-1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, inactivation NIK, SAP K, SAP-1, JNK, ifn response gene, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK part, Ox40, Ox40 part, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP1 or TAP2 or its function fragment.

B. route of administration

The vaccine can be applied by different approach, the approach include orally, parenteral, Sublingual, percutaneous, Jing Rectum, mucosa, partly, apply via suction, via cheek, in thoracic cavity, intravenouss, intra-arterial, intraperitoneal, subcutaneous, intramuscular, Nose is intracapsular and intraarticular or its combination.For veterinary uses, the combination can be according to the veterinary code of standard as suitable Apply when acceptable preparation.Veterinary can determine the dosage regimen and route of administration for being best suited for particular animals easily.Institute Stating vaccine can be by traditional syringe, needleless injection device, " microparticle bombardment rifle " or other physical methods such as electroporation (" EP "), " hydrodynamic method " or ultrasound wave are applying.

The carrier of vaccine can be delivered to mammal by some well known technologies, and the technology has been included in and has not had There are DNA injections in the case of the recombinant vector that In vivo electroporation, liposome-mediated nano-particle promote (also referred to as DNA epidemic diseases Seedling is inoculated with), the recombinant vector such as recombinant adenoviruss, recombinant adeno-associated virus and recombinant vaccinia.The HBV antigens can Can inject via DNA and be delivered in company with internal electroporation together.

C. electroporation

Apply the vaccine to complete using electroporation device via the electroporation of the plasmid of vaccine, the electricity is worn Hole equipment is configured to the pulse energy for being effectively formed reversible pore to required mammalian tissues delivering in cell membrane, And preferably described pulse energy is the constant current similar to the electric current set in advance of user input.The electroporation Equipment can include electroporation part and electrode assemblie or Handleset.Electroporation part can include and be incorporated to electroporation device Various elements in one or more, which includes：Controller, current waveform generator, impedance measuring instrument, curve tracer, Input element, status reporting element, COM1, memory unit, power supply and on and off switch.Electroporation can be using electricity in vivo Perforating apparatus such as CELLECTRAEP systems (Yin Nuowei pharmaceutical Co. Ltds (Inovio Pharmaceuticals, Inc.), Lan Ling cities (Blue Bell), Bin Nifaniya states (PA)) or Elgen electroporation apparatuss (the limited public affairs of Yin Nuowei pharmacy Department) complete promoting plasmid-transfected cells.

The electroporation device of delivering and the example of electroporation method of the DNA vaccination of the present invention can be promoted to be included in De La The U.S. Patent number 7,245,963 of Ji Ya-Oakley (Draghia-Akli) etc., by the U.S. of the submissions such as Smith (Smith) Patent discloses those described in 2005/0052630, and their content is integrally incorporated herein by reference.Can be used for promoting Other electroporation devices and electroporation method for entering the delivering of DNA vaccination are included in the co-pending of the submission of on October 17th, 2007 And jointly owned United States Patent (USP) discloses provided in serial number 11/874072 those, the patent is disclosed according to the U.S. The 35th the 119th article of (e) money of code requires the U.S.Provisional Serial 60/852,149 submitted on October 17th, 2006 The rights and interests of the U.S.Provisional Serial 60/978,982 submitted to on October 10th, 2007, all of which are integrally incorporated this Text.

The U.S. Patent number 7,245,963 of De Lajiya-Oakley etc. describes Modular electrical electrode systems and they use Use in promoting to introduce biomolecules into body or plant in the cell of selected tissue.The Modular electrical electrode systems Multiple needle electrodes can be included；Hypodermic needle；There is provided from programmable constant current pulses controller to multiple needle electrodes The electric connector of conductivity connection；And power supply.Operator can catch the multiple needle electrodes fixed on the support structure and incite somebody to action They are firmly inserted in selected tissue in body or plant.Then biomolecule delivery is arrived via hypodermic needle In selected tissue.Start programmable constant current pulses controller, and constant current electric pulse is applied to into multiple pins In electrode.During the constant current electric pulse applied promotes the cell introduced biomolecules between multiple electrodes.The U.S. is special The full content of profit number 7,245,963 is incorporated herein by reference.

The United States Patent (USP) of the submissions such as Smith discloses 2005/0052630 and describes to can be used to be effectively facilitated biological point Son selectes the electroporation device in the cell of tissue in being incorporated into body or plant.The electroporation device includes its operation by soft Part or firmware are come the electrodynamics equipment (" EKD equipment ") that specifies.The EKD equipment is based on user control and pulse parameter It is input into and a series of programmable constant current pulses pattern is produced between the electrode of an array, and allows current waveform The storage and acquisition of data.The electroporation device also includes the interchangeable electrode discs with a pricking with needle electrode, for noting Penetrate the central injection canal and removable guide disk of pin.United States Patent (USP) discloses 2005/0052630 full content to quote Mode is incorporated herein.

In U.S. Patent number 7,245,963 and United States Patent (USP), electrod-array and method described in 2005/0052630 are disclosed Can be adapted to for being not only penetrate deep into organizing as in muscle, and be penetrate deep in other tissues or organ.By In the configuration of electrod-array, injection needle (to deliver the biomolecule of selection) is also fully inserted in target organ, and is injected It is on the target tissue being vertically applied in the region described by these electrodes in advance.In 7,245,963 He of U.S. Patent number United States Patent (USP) discloses these electrodes described in 2005/005263 and is preferably 20mm length and No. 21 specifications.

Additionally, what is covered in some embodiments is the electroporation device and its use for merging, there is electroporation device to retouch State in following patent：The U.S. of the United States Patent (USP) bulletin on the 29th of August in 5,273,525,2000 of the bulletin of on December 28th, 1993 Patent 6,110,161, on July calendar year 2001 17 bulletin 25,6,261,281 and 2005 on October bulletin 6,958,060 with And the United States Patent (USP) 6,939,862 of the bulletin on the 6th of September in 2005.Additionally, covering the patent for covering theme, the theme herein Any one provided in plurality of devices is directed to use with carrys out the United States Patent (USP) 6,697 of on 2 24th, 2004 bulletins of DNA delivery, 669 and with regard to injecting in the United States Patent (USP) 7,328,064 of on 2 5th, 2008 of method bulletins of DNA.The whole of above patent Content is herein incorporated by reference.

D. the method for preparing vaccine

Provided herein is for the method for preparing the DNA plasmid comprising DNA vaccination discussed in this article.The DNA plasmid exists After final subcloning steps enter Mammalian expression plasmid, can be used for the side known in the art used in large fermentation tank Method inoculating cell culture.

DNA plasmid for being used together with the EP equipment of the present invention can be matched somebody with somebody using the combination of known device and technology System is manufactured, it is preferred that they are manufactured using optimization plasmid manufacturing technology, the technology is described on May 23rd, 2007 In the application number 20090004716 of the U.S. Publication of submission.In some instances, the DNA plasmid used in these researchs can Prepare with by the concentration more than or equal to 10mg/mL.Manufacturing technology also includes or to be incorporated to those of ordinary skill in the art universal Known various equipment and scheme, in addition to those described in United States serial 60/939792, also including on July 3rd, 2007 Those described in the license patent of bulletin, U.S. Patent number 7,238,522.Above-cited application and patents, U.S.'s sequence Number 60/939,792 and U.S. Patent number 7,238,522 full content be respectively incorporated into herein.

Embodiment

The present invention is further illustrated in the examples below.It will be appreciated that these embodiments are in the excellent of the expression present invention Only by explanation being given when selecting embodiment.From discussion above and embodiment, those skilled in the art can be true The necessary characteristic of the fixed present invention, and the various change of the present invention without departing from the spirit and scope, can be made With modification to adapt it in various usages and condition.Therefore, except shown and described herein in addition to those, from the above description The various modifications of the present invention will be apparent to those skilled in the art.These modifications are also intended to belong to appended power In the range of profit is required.

Total HBcAg be also referred to as HBV modification or M- core constructs, it be from from HBV gene type A, Design in the epitope sequences of B, C, D and E.The HBcAg sequence from these genotype is selected being included in total core In the structure of the heart, the total core can induce the immunity for broad range of genotype, so as to provide the general epidemic disease of HBV Seedling.In some embodiments, the modification of M- core constructs includes adding IgE targeting sequencings.In some embodiments, M- Core protein is to optimize to encode using the codon optimization for Enhanced expressing and RNA.

By coding with IgE targeting sequencings and HA labellings (SEQ ID NO:5) the nucleotide sequence clone of M- core sequences To in expression vector pVAX to produce construct pM-core.Vivoexpression is tested using pM constructs and pVAX to complete simultaneously And it is used as control.During the gel images that the result of positive expression is illustrated in being depicted in Fig. 2A and Fig. 2 B are shown.

C57BL/6 transgenic mices be divided into two groups of per group of 4 mices and using electroporation come with the DNA of 20 μ g by every Three (group 1-pVAX vehicle Controls of Immunity at intervals biweekly；Group 2pM-core).Mice was at the 0th day, the 14th day, the 28th day It is condemned to death by immunity and at the 35th day.Spleen, liver and serum are harvested from the animal put to death.

The In vivo study of C57BL/6 mouse species shows from spleen the tumor necrosis factor in CD8 the and CD4 T cells for obtaining The amount of the secretion of sub (TNF-α), interferon gamma T- cells (IFN-γ) and CD 107a increases.Fig. 3 A and Fig. 3 B show and use pM- Core carries out the amount of IFN-γ secretion of the C57BL/6 mices of vaccination in CD8+ the and CD4+T cells of spleen to be increased. Fig. 4 A and Fig. 4 B show TNF-α secretion amount increase.Fig. 5 A and Fig. 5 B show The amount of the CD 107a secretions in dirty CD8+ and CD4+T cells increases.

Migration of the HBV specific T-cells to liver is also demonstrated in the animal for applying pM-Core DNA vaccinations.With height It is the important goal for researching and developing HBV immunization therapies that frequency and the effector function to liver carry out targeting HbcAg specific T-cells. After immunity, put to death animal and remove their liver, and determine migration of the sub- T cell of HBV specific effectors to liver.Knot Fruit shows that pM-Core vaccines are driven to effector T cell in liver in vivo.Fig. 6 A and Fig. 6 B illustrate interferon-γ T cell Liver response, Fig. 7 A and Fig. 7 B illustrate the immunoreation of tumor necrosis factor-alpha liver and are drawn by vaccination is carried out with pM-Core The reaction of the raising for rising.

The CD4+/CD8+T cells of strong balance are driven by the total immunogen of M- cores that pM-core DNA construct is encoded Immunoreation.The T cell induced after HBV infection is passed in liver and is showed for the correct of immune clearance with altofrequency Effector phenotype.

Fig. 8 to show and determine anti-by the cellular immunization of pM-Core inductions using Enzyme-linked Immunosorbent Assay speckle (ELISPOT) Should.Stimulate splenocyte with two set of the 15-mer peptides overlapped across whole pMCore length and with 8 aminoacid.Will 200,000 splenocytes in R10 culture medium are seeded in IFN-γ capture antibody (R＆D systems (R＆D system)) in 96 holes and apply In the plate of cloth, and the 5%CO at 37 DEG C₂In stimulate overnight in the presence of specific peptide set.Cell is rinsed out and is made plate and life Anti-mouse IFN-γ detection antibody (R＆D systems) overnight incubation of thing elementization.Subsequently by Streptavidin-alkali phosphatase and 5- The chloro- 3 '-indolyl phosphate para-totuidine salt of bromo- 4- and NBT are used to grow speckle.Using automatization ELISPOT readers (CTL company limiteies (CTL Limited)) count speckle.As shown in figure 8, can be lured with pMCore immunity Lead strong cell immune response.Data display Dominant Epitopes deflection peptide set 2.Average HBcAg- specificity IFN-γ T cells Reaction is every million splenocyte about 2000 (± 210) SFU.

In vivo cytotoxicity determines research using the CF 5(6)-Carboxyfluorescein oxalic acid butanimide combined with flow cytometry Ester (CFSE) labelling is carrying out.We have evaluated the cell division in the cell of cell mass.By splenocyte from the little of initial experiment Two groups are separated and are classified as in Mus.One group of the CFSE numbers of altitude is processed with relevant peptide (such as HBV core peptides) pulse. Another group of CFSE minuent labellings is processed with incoherent peptide (such as HCV NS3 peptides) pulse.Will be labelling, peptide process thin During born of the same parents combine and the adoptive transfer for carrying out flow point analysis is tested.The combination group of the target cell of process, labelling is administered to Two groups of mices, i.e. matched group and immune group.From separating Morr. cell in every group of mice, and sample is run on flow cytometer. The quantity of measurement CFSE.Generally, in such experiment, two peaks are formed, first is uncorrelated peptide；Second is to indicate more Immune peptide in the peak of big CFSE.

Fig. 9 to be illustrated and can clearly remove internal target cell by the CD8 T cells induced by vaccination.These results Show the spleen of mice and the sample of liver from initial experiment contain in the peak of uncorrelated peptide and related peptides almost etc. The cell of amount, while these results are clearly illustrated in immune group, from the cell of those for entering horizontal pulse with related peptides Peak significantly less than incoherent peptide.The target cell that these data displays HBV peptides are processed is carrying out the little of immunity with HBV vaccines Clearly removed in Mus, but do not had in non-immune mice.Any removing of the target cell processed with uncorrelated peptide is such as Fruit all occurs, then which is identical in the mice that immunity is carried out with HBV vaccines and in non-immune mice, and significantly Less than the removing of the target cell processed with HBV peptides.

Figure 10 illustrates from the T cell proliferation assay using CFSE labellings the data collected.It is relatively (more right with pVax carriers According to) or with expression HBV M- cores plasmid pMCore process CD3+CD4+ cells and CD3+CD8+ propagation percentage ratio. Briefly, the explanation according to manufacturer, by detached splenocyte CF 5(6)-Carboxyfluorescein oxalic acid succinimide ester (CFDA- SE) Cellular tracking agent test kit (Cell Tracer Kit) (hero company) is dyeing.Staining cell normal saline washing three times is simultaneously Stimulated with pMCore- specificitys overlapping peptide.Cell is made to be incubated 96 hours at 37 DEG C.After 48 hours, the culture medium of removing 50% is simultaneously Changed with fresh R10.At the 4th day, harvesting was simultaneously used CD3, CD4 and CD8- monoclonal antibody specific (BD Pharmingen) dye.Cell is fixed and at flow cytometer (FACScalibur) with the PBS containing 1% paraformaldehyde (PFA) The cell is obtained on (Bi Di Medical Devices Co., Ltd.s (Becton Dickinson)).Number is analyzed using FlowJo programs According to.What CFSE was low is considered as the cell of propagation with CFSE medium group.As shown in Figure 10, it is isolated from the CD3+CD8+T of spleen Cell is bred more compared with CD3+CD4+T cells.

Figure 11 A and Figure 11 B are illustrated come pVax carriers (control) or the plasmid pMCore with expression HBVM- cores of using by oneself The ELISA data of the comparison of anti-HBV core antibodies in the serial dilutions of serum of the animal of process.Briefly, by high combination Elisa plate (Corning Incorporated (Costar), healthy and free from worry (Coming), New York (NY)) is applied with 1 μ g/ml HBcAg protein in PBS Cloth, places 24h at 4 DEG C, and and then rinsed with PBS- tweens, and close 2h at room temperature with the PBS containing 1%BSA.Will The serum sample of serial dilution is added in hole, and is incubated 1h at room temperature.After flushing, resisted by the goat of HRP- labellings little Mus IgG (Figure 11 A) or IgA (Figure 11 B) is showing the serum antibody of combination.Using tetramethyl benzidine (Sigma Ao Ruiqi Company (Sigma-Aldrich)) as substrate detecting the Ab of peroxidase conjugated, and read with multiple scaaning elisa plate Take device to measure the OD at 450nm.Antigen-specific humoral reaction of the observation in the serum collected from immune mouse.

Figure 12 shows TNF-α and IFN-γ percentage ratio in CD4+ and CD8+ spleen and liver cells.

In the case of the small animal model that there is no HBV, being injected by hydrodynamics is used for transient transfection by HBcAg Mouse liver.The mouse liver of immunity is transfected with pMCore or HCV NS3/4A.Three days immunohistochemical stainings after transfection Show the hepatocellular removing of the HBcAg- transfections compared with the hepatocyte of NS3/4A- transfections.Measurement serum in ALT levels so as to Guarantee that the removing induced by immune mouse does not cause any hepatic injury.Result in Figure 13 is shown by the clear of immune mouse induction Except not causing any hepatic injury.

Claims (20)

1. a kind of nucleic acid molecules, the nucleic acid molecule encoding induce the sequence of the total HBcAg of immunoreation in individuality Arrange, the albumen is：SEQ ID NO:2、SEQ ID NO:4 or SEQ ID NO:6 protein.

2. nucleic acid molecules as claimed in claim 1, which further includes a nucleotide sequence, and the sequential coding is connected to institute State the signal peptide of the N-terminal of protein.

3. nucleic acid molecules as claimed in claim 1, its coding SEQ ID NO:2 protein.

4. nucleic acid molecules as claimed in claim 1, its nucleotide sequence is SEQ ID NO:1.

5. nucleic acid molecules as claimed in claim 1, which further includes a nucleotide sequence, and the sequential coding is connected to institute State the 5 ' of the protein sequence signal peptide held.

6. nucleic acid molecules as claimed in claim 1, its nucleotide sequence be selected from group consisting of one or more Nucleotide sequence：SEQ ID NO:1；SEQ ID NO:3；And SEQ ID NO:5.

7. nucleic acid molecules as claimed in claim 1, wherein the nucleic acid molecules are incorporated in virion.

8. a kind of plasmid, it is characterised in that the plasmid includes the nucleic acid molecules described in claim 1.

9. a kind of expression vector, it is characterised in that the expression vector includes the right for being operatively connected to controlling element Require the nucleic acid molecules described in 1.

10. the purposes of nucleic acid molecules as claimed in claim 1, it is characterised in that for preparing induction exempting from for HBV antigens The medicine of epidemic disease reaction.

The purposes of 11. nucleic acid molecules as claimed in claim 1, it is characterised in that protect individuals from HBV for preparing one kind The medicine of infection.

The purposes of 12. nucleic acid molecules as claimed in claim 1, it is characterised in that be diagnosed trouble for preparing a kind of protection There is the medicine of the individuality of HBV infection.

A kind of 13. SEQ ID NO:2 protein.

A kind of 14. SEQ ID NO:4 protein.

A kind of 15. SEQ ID NO:6 protein.

The purposes of 16. protein as described in claim 13-15 is arbitrary, it is characterised in that be directed to for preparing a kind of induction The medicine of the immunoreation of HBV antigens.

The purposes of 17. protein as described in claim 13-15 is arbitrary, it is characterised in that individual for preparing a kind of protection From the medicine of HBV infection.

The purposes of 18. protein as described in claim 13-15 is arbitrary, it is characterised in that for prepare a kind of protection by The medicine of individuality of the diagnosis with HBV infection.

A kind of 19. vaccines suitable for the immunoreation for HBV is produced in subject, the vaccine are included：

Nucleic acid molecules according to claim 1, and

Adjuvant molecules.

20. vaccines as claimed in claim 19, wherein the adjuvant is IL-12, IL-15, IL-28 or RANTES.